Short-term improvements in retinal anatomy are known to occur in preclinical models of photoreceptor transplantation. However, correlative changes over the long term are poorly understood. We aimed to develop a quantifiable imaging biomarker grading scheme, using noninvasive multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, to enable serial evaluation of photoreceptor transplantation over the long term. Methods: Photoreceptor cell suspensions or sheets from rhodopsin-green fluorescent protein mice were transplanted subretinally, into either NOD.CB17-Prkdc scid /J or C3H/HeJ-Pde6b rd1 mice. Multimodal cSLO imaging was performed serially for up to three months after transplantation. Imaging biomarkers were scored, and a grade was defined for each eye by integrating the scores. Image grades were correlated with immunohistochemistry (IHC) data. Results: Multimodal imaging enabled the extraction of quantitative imaging biomarkers including graft size, GFP intensity, graft length, on-target graft placement, intra-graft lamination, hemorrhage, retinal atrophy, and periretinal proliferation. Migration of transplanted material was observed. Changes in biomarker scores and grades were detected in 14/16 and 7/16 eyes, respectively. A high correlation was found between image grades and IHC parameters. Conclusions: Serial evaluation of multiple imaging biomarkers, when integrated into a per-eye grading scheme, enabled comprehensive tracking of longitudinal changes in photoreceptor cell grafts over time. The application of systematic multimodal in vivo imaging could be useful in increasing the efficiency of preclinical retinal cell transplantation studies in rodents and other animal models. Translational Relevance: By allowing longitudinal evaluation of the same animal over time, and providing quantifiable biomarkers, non-invasive multimodal imaging improves the efficiency of retinal transplantation studies in animal models. Such assays will facilitate the development of cell therapy for retinal diseases.

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Purpose Cell-based therapy development for geographic atrophy (GA) in age-related macular degeneration (AMD) is hampered by the paucity of models of localized photoreceptor and retinal pigment epithelium (RPE) degeneration. We aimed to characterize the structural and functional deficits in a laser-induced nonhuman primate model, including an analysis of the choroid. Methods Macular laser photocoagulation was applied in four macaques. Fundus photography, optical coherence tomography (OCT), dye angiography, and OCT-angiography were conducted over 4.5 months, with histological correlation. Longitudinal changes in spatially resolved macular dysfunction were measured using multifocal electroretinography (MFERG). Results Lesion features, depending on laser settings, included photoreceptor layer degeneration, inner retinal sparing, skip lesions, RPE elevation, and neovascularization. The intralesional choroid was degenerated. The normalized mean MFERG amplitude within lesions was consistently lower than control regions (0.94 ± 0.35 vs. 1.10 ± 0.27, P = 0.032 at month 1, 0.67 ± 0.22 vs. 0.83 ± 0.15, P = 0.0002 at month 2, and 0.97 ± 0.31 vs. 1.20 ± 0.21, P < 0.0001 at month 3.5). The intertest variation of mean MFERG amplitudes in rings 1 to 5 ranged from 13.0% to 26.0% in normal eyes. Conclusions Laser application in this model caused localized outer retinal, RPE, and choriocapillaris loss. Localized dysfunction was apparent by MFERG in the first month after lesion induction. Correlative structure-function testing may be useful for research on the functional effects of stem cell–based therapy for GA. MFERG amplitude data should be interpreted in the context of relatively high intertest variability of the rings that correspond to the central macula. Sustained choroidal insufficiency may limit long-term subretinal graft viability in this model.

32Purpose: Short-term improvements in retinal anatomy are known to occur in preclinical models 33 of photoreceptor transplantation. However, correlative changes over the long term are poorly 34 understood. We aimed to develop a quantifiable imaging biomarker grading scheme, using non-35 invasive multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, to enable serial 36 evaluation of photoreceptor transplantation over the long term. 37Methods: Yellow-green fluorescent microspheres were transplanted into the vitreous cavity 38 and/or subretinal space of C57/BL6J mice. Photoreceptor cell suspensions or sheets from 39 rhodopsin-green fluorescent protein mice were transplanted subretinally, into either NOD.CB17-40 Prkdc scid /J or C3H/HeJ-Pde6b rd1 mice. Multimodal cSLO imaging was performed serially for up 41 to three months after transplantation. Imaging biomarkers were scored, and a grade was defined 42 for each eye by integrating the scores. Image grades were correlated with immunohistochemistry 43 (IHC) data. 44 Results: Multimodal imaging enabled the extraction of quantitative imaging biomarkers 45 including graft size, GFP intensity, graft length, on-target graft placement, intra-graft lamination, 46 hemorrhage, retinal atrophy, and peri-retinal proliferation. Migration of transplanted material 47 increasing the efficiency of preclinical retinal cell transplantation studies in rodents and other 53 animal models. 54 Key words: degenerative retinal diseases, age-related macular degeneration, stem cell therapy, 55 xenotransplantation, photoreceptor cell, retinal organoid, confocal scanning laser 56 ophthalmoscopy 57 Photoreceptor transplantation is being developed as a therapeutic modality to restore vision in 59 people affected by retinal degenerative diseases, including retinitis pigmentosa (RP) and age-60 related macular degeneration (AMD) 1-6 . Short term improvements in outer retinal anatomy after 61 photoreceptor cell transplantation have been observed, mainly by histological staining in 62 preclinical models of retinal degeneration 4,7 . However, histology is a relatively inefficient 63 method to track graft and recipient anatomy longitudinally over the long term. Histological 64 assays are labor-intensive, require large initial cohorts of recipients, and face the challenge of 65 recipient attrition over time. Non-invasive imaging could facilitate the longitudinal evaluation of 66 retinal anatomy in relatively smaller cohorts of recipient animals over time, without the need to 67 sacrifice animals at every assessment time point. 68 Recent advances in imaging techniques have enabled detailed imaging studies in mouse models 69 of retinal disease and regeneration 8-10 . Confocal scanning laser ophthalmoscopy (cSLO) can be 70 used to capture images in multiple imaging modes, including short-wavelength fluorescence 71 (SWF) excitation (488 nm) to detect photoreceptor cells labeled with green fluorescent protein 72 (GFP) in mouse recipients 1 . Multicolor reflectance (MR) imaging combines blue (488 nm)...

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Human retinal organoid transplantation can potentially restore vision in patients with degenerative retinal diseases. How the recipient retina regulates the maturation, fate specification, and migration of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice, conducted histology and single-cell RNA sequencing analyses, and observed two main classes of graft-derived cells. The first class consisted of retinal astrocytes and brain/spinal cord-like neural precursors, absent or rare in cultured organoids, that migrated into all recipient retinal layers and traveled long distances. The second class consisted of retinal progenitor-derived cells, including rods and cones, that remained in the subretinal space and matured more rapidly than photoreceptors in culture. These data suggest that the recipient subretinal space promotes the maturation of transplanted photoreceptors while inducing or expanding migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for cell-based treatment of retinal diseases.